Histology Mentor

Cytology, Methods and Epithelia, Questions 1 - 15

1. Question - Diagram the structure of biological membranes remembering that intracellular membranes have the same type of structure seen in the plasma membrane. Illustrate four processes that occur at plasma membranes.

Biological membranes consist of two lipid layers creating a central hydrophobic region. Polysaccharides are only associated with the outer surface of the plasma membrane. Freeze-fracture of the plasma membrane separates the two phospholipid bilayers and reveals the distribution of proteins in the membrane. The fracture plane preferentially follows the lipid-lipid interface of membranes and the associated proteins appear as bumps on the surface. The outer surface of the inner leaflet is called the P face and displays the greatest number of particles. The inner surface of the outer leaflet is called the E face.

2. Question - Diagram and label features associated with a mitochondrion.

3. Question - What is a lysosome? Trace, with a diagram, the formation of a primary lysosome beginning with peptide synthesis. What are ribosomes and polyribosomes? How do attached and free polysomes differ in function?

Lysosomes are membrane limited vesicles containing hydrolases having an acid pH optimum. They merge with endosomes and autosomes to form secondary lysosomes in which the contents are acidified and digested. Protons are pumped from the cytosol into the vesicle by a membrane-bound vesicular proton ATPase.

Ribosomes are the machinery for interpreting messenger RNA (mRNA) to put together amino acids to form peptides. Usually a single mRNA molecule will have several ribosomes working on it at once. This means that those that have just started the process will have short segments of peptides attached to them and those that are nearly finished will have long segments. A group of ribosomes attached to a single mRNA is called a polysome or polyribosome. Polysomes can either be attached to endoplasmic reticulum or they can be free in the cytoplasm. The attached ones are making proteins that will be sequestered in membrane-limited vesicles (lysosomes or secretion granules) while the free ones make proteins for the cytoplasm.

4. Question - Diagram a nucleus and its envelope indicating the function of each component. Name the phases of the mitotic cycle. What is pyknosis?

Resting cells that are not mitotically active are said to be in G-zero phase. G-one (G₁) is the phase in which the cell is tooling up for S phase, DNA synthesis. G-two includes the events that prepare for mitosis (M phase). Mitosis is divided into 4 phases - prophase, metaphase, anaphase and telophase. Pyknosis is the process in which the nucleus becomes very small and compact. It frequently indicates a dying cell.

5. Question - Describe the molecular composition of microtubules and cytoplasmic filaments? How do they serve the cell activities?

Microtubules are tubular nonbranching structures assembled from two different globular proteins, alpha and beta tubulin. They are about 22 nm in diameter and provide a "monorail" on which cell components are moved to (dynein) and from (kinesin) the cell center, the centrioles. Kinesin and dynein are ATP dependent "motors" that link elements to be transported to the micortubules. Each centriole consists of 9 triplets of short microtubules. They function to organize the mitotic spindle apparatus that consist of microtubules. Microtubules are also involved in maintaining cell shape and ciliary and flagellar movements.

Actin occurs in two forms globular (g-actin) and fibrillar (f-actin). F-actin is assembled from g-actin forming the smallest cytoplasmic filaments, about 8 nm in diameter. Besides being involved in cellular contraction actin moves cell processes, participates in endo- and exocytosis and cellular locomotion.

Intermediate filaments

 

6. Question - What are cytoplasmic inclusions? Describe their structure and explain their function.

Glycogen is a polymer of glucose and forms clusters of granules in the cytoplasm without a surrounding membrane. It is a storage form of glucose.

Fat occurs as droplets in the cytoplasm and that are now known to be surrounded by a single leaflet of a biological membrane. The membrane contains enzymes that facilitate the deposition and mobilization of the fat stored there. The lipid can also be used intracellularly as a source of energy or used as synthetic precursors.

Lipofuscin is a pigment granule that contains nondigestible debris of lysosomal activity. It is membrane limited and is also called aging pigment or tertiary lysosome.

Secretion granules and melanosomes are also called inclusions.

 

7. Question - What are the basic steps in preparing a tissue for microscopic examination?

Fixation - stops enzyme activity and postmortem changes and immobilizes cellular constituents. It can be accomplished by various solutions, 4% formaldehyde being the most common.

Dehydration - removes water from tissues by replacing it with alcohol

Clear - replaces water soluble solvent with paraffin soluble solvent

Infiltrate - replaces solvent with polymer usually paraffin

Embed - tissue is oriented and paraffin allowed to solidify

Section - blocked tissue is cut into thin sections about 5 micrometers thick using a microtome and placed on slide

Stain - Staining involves removal of paraffin from the section, treatment with stains, and finally dehydration and putting a coverslip on the section with a plastic mounting medium.

 

8. Question - What are the components of a routine histological stain? What are their chemical characteristics and what do they stain? What stains are used to demonstrate polysaccharides, elastic fibers and reticular fibers?

Hematoxylin is a cationic, blue or purple stain and tends to bind to anionic polymers such as nucleic acids and acidic polysaccharides. It is said to be a basic stain and the components that take hematoxylin are said to be basophilic.

Eosin is an anionic, pink stain. It tends to bind to cationic polymers such as proteins in an acid environment. It is said to be an acid stain and the components that stain with it are said to be acidophilic or eosinophilic.

The periodic acid Schiff stain (PAS) demonstrates polysaccharides such as in glycogen, mucus, and basement membranes.

Orcein stains elastic fibers and a silver stain turns reticular fibers black.

 

9. Question - What is a tissue? What are the fundamental characteristics of the four basic tissue types? In which of these is cell renewal an important process?

A tissue is a collection of similarly differentiated cells that cooperate with each other for the performance of a specific function. Every organ can be described in terms of the types, amounts and arrangements of its component tissues. Four classes of tissues are recognized; epithelia, connective tissue, muscle and nerve.

Epithelia consist of cells closely associated with each other (little intercellular material and capillaries are typically not present between the cells; nerve fibers can be there). Surface epithelia and epithelia that form exocrine glands occur in continuous layers so that they separate environments having different compositions.

Connective tissue is the supporting tissue and consists of much intercellular matrix (fibers, ground substance and fluid). Fixed and wandering cells as well as blood vessels and nerves are found in connective tissues.

Muscle consists of contractile cells harnessed by connective tissue carrying blood vessels and nerves.

Nerve tissue consists of nerve cells and or their processes. In the peripheral nervous system connective tissue provides support. In the central nervous system support is provided by glial cells. Myelin occurs in segments along nerve processes and is made by Schwann cells in the PNS and by oligodendrocytes in the CNS

Surface epithelia are constantly being renewed but in other places such as the liver little proliferation occurs unless it is injured. Connective tissue rapidly regenerates when injured.

 

10. Question - How are surface epithelia classified? Diagram examples of each.

Epithelia are classified on the basis of the number of cell layers (one layer is called simple and multiple layers is called stratified) and on the shape of the surface cells (squamous, cuboidal and columnar).

Some special epithelia are: Endothelium is simple squamous epithelium lining all vascular channels, blood and lymph. Mesothelium is simple squamous epithelium that lines serous cavities, the peritoneal, pleural and pericardial cavities. In Pseudostratified epithelium all the cells rest on the basement membrane but only some reach the surface. In Transitional epithelium (urothelium) the number of cell layers appears to change with distension. The surface cells are especially adapted to accommodate great changes in the surface area. In stratified squamous epithelia the surface cells die (indicated by dark, dense, pyknotic nuclei) and are either sloughed off or form keratin.

11. Question - Compare and contrast exocrine glandular epithelia with surface epithelia. Diagram the cytology of a serous acinar cell.

Both consist of cells closely associated with each other. Glandular epithelia usually form tubules or acini while surface epithelia form sheets. But some surface epithelia contain glandular cells, both exocrine and endocrine. Secretion by cells that store their secretions in secretion granules is said to be regulated whereas cells that secrete continuously are said to do so constitutively.

 

12. Question - Make a drawing(s) that illustrates how epithelial cells are attached to each other and to supporting tissue. What is the composition of the basement membrane?

By light microscopy the junctional complex is seen as a dot and is called the terminal bar. It is resolved into at least 3 components by electron microscopy, the zonula occludens that provides a seal and for physical attachment the zonula adherens and macula adherens. The basement membrane is a layer of extracellular matrix as seen with the light microscope. It consists of at least two regions that are identifiable with the electron microscope.

 

1) A relatively amorphous layer only resolved with EM called the basal lamina. Some of its components are type IV collagen, heparan sulfate, fibronectin and laminin. It is usually separated from the epithelium by an electron clear band (lamina rara) thought to be bridged by laminin and fibronectin. These molecules have sites that bind more or less tightly to collagen, heparan and integrins that span the cell membrane.

2) The reticular lamina is a rather thick layer of reticular fibers (type III collagen) in an amorphous matrix. It is produced by the connective tissue and is argyrophilic (Reticular fibers stain with silver stains.).

 

13. Question - Illustrate the structure of microvilli and cilia; how do they differ in function? How are they anchored in the cytoplasm?

The microvillus is primarily specialized for absorption although, since they contain actin filaments, some movement (extension and contraction) may occur. The cilium has a beating motion designed for moving fluids along the surface but some absorption may occur. Ciliary motion in a plane perpendicular to the plane of the central pair of microtubules occurs when microtubule doublets interact via dynein arms.

14. Question - Recall and define 13 terms that are used to describe and explain the structure and function of exocrine glands. How does and exocrine gland differ from an endocrine gland?

Eccrine secretion indicates secretion through exocytosis

Apocrine secretion is the process in which a portion of cytoplasm is pinched off including some plasma membrane.

Holocrine secretion is the process in which the cell containing the secretion product dies and is pushed out of the gland as more cells accumulate and die.

Simple gland is one in which the duct is not branched.

Compound gland is one in which the duct is branched.

Tubular gland is one in which the secretory portion forms a tube usually mucus-secreting.

Acinar gland contains spherical secretory units, acinii. Alveolus is often used as a synonym but strictly describes an oval structure with a larger lumen than an acinus.

Tubulo-alveolar gland contains a mixture of morphological secretory units.

Serous demilune is a group of serous cells capping over the end of a mucous tubule.

Serous gland or cell secretes a watery product.

Mucous gland or cell secretes mucus, a very viscous product.

Stroma is the supporting tissue of an organ. It usually consists of connective tissue. This can be applied to any organ not just exocrine glands.

Parenchyma is the cells that perform the specialized functions of the organ. In the case of exocrine glands this would comprise the acini and the ducts.

Endocrine glands are composed of epithelial cells that produce hormones. Epithelial cells are organized in small clumps, irregular cords, irregular plates, or follicles surrounded by basal lamina and closely associated with numerous fenestrated capillaries. There are no ducts.

 

15. Question - The submandibular gland is an example of a compound tubulo-alveolar gland. Explain what that means and make a line drawing illustrating 5 features that characterize such a gland. What are myoepithelial cells?

Glands with an unbranched duct system are said to be simple; compound glands have a branched duct system. The descriptors tubulo-alveolar refer to the shapes of the secretory components of the gland. Alveolar and acinar are often used interchangeably. Myoepithelial cells are contractile cells that have many processes that surround the secretory tubules and acini within their basement membranes.

 

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